The amelogenins are extracellular matrix proteins, produced by ameloblasts during tooth enamel formation. These proteins are unique to enamel and are required for normal development of enamel mineral. The purpose of this application is to investigate regulation of expression of the X- chromosomal amelogenin gene, by analyzing the 3.5 KB fragment able to direct expression of a reporter gene to ameloblasts in transgenic mice, by sequence analysis and testing selected segments for activity using transgenic mice. We will also create in vivo a murine null mutation for the amelogenin gene, and express leucine rich amelogenin protein (RAP) in transgenic mouse enamel to determine whether overexpression will interfere with the normal function of amelogenin proteins. Mating of the knockout and LRAP mice will yield offspring which secrete LRAP as the only amelogenin protein in developing enamel. The specific aims are: (1) to characterize the regulatory sequences of the X-chromosomal amelogenin gene, using transgenic mice; (2) to construct a "knockout" mouse for the X-chromosomal amelogenin gene; (3) to construct a transgenic mouse that overexpresses LRAP in ameloblasts under control of the amelogenin regulatory sequence; and (4) to replace the amelogenin protein with LRAP in mouse tooth enamel. In these experiments, teeth from the 3 genetically altered mouse lines will be compared histologically for tissue alterations, and for quality and composition of mineral crystal, in order to better understand the role of the organic matrix in the mineralization process.